This grant application addresses broad challenge area (taken directly from the NHLBI description of RFA-OD-09-003: NHLBI Challenge Grants. We have underlined the portions of this Challenge Grant relevant to this application.) "06: Enabling technologies, Challenge Topic: 06-HL-109: Generate reagents for studying lung cell biology and disease progression. Reagents for studying lung cell biology and disease progression are lacking. Examples include antibodies that recognize specific cell types, promoters that are expressed only in certain cell types and can be used in the generation of conditional knockout transgenic animals, and antibodies that recognize cell surface markers and can be used for FACS sorting different cell lineages in the airway. Such markers would be important not only for understanding the heterogeneity of lung cell types but also for understanding cellular changes in the lung that emerge with lung disease. They may also be useful as surrogates for progression of lung disease and for dissecting cellular heterogeneity/function of lung cell types." The pulmonary alveolar epithelium, which covers >99% of the internal surface area of the lung, is comprised of two types of cells, type I and type II cells, both of which are believed to be essential for mammalian life. Type I cells are very large squamous cells with calculated diameters of 50-100 [unreadable]m;the very thin (~50 nm) cytoplasmic extensions of the Type I cells cover the basement membrane separating the epithelial from the interstitial and vascular compartments. Type II cells are smaller, cuboidal cells (diam. ~10 [unreadable]m) characterized morphologically by secretory granules called lamellar bodies. Although the functions of type I cells are less well understood than the functions of type II cells, type I cells are believed to play an important role in the lung because they cover more than 98% of the internal surface area of the lung, providing a narrow anatomic barrier between the air and blood compartments critical for efficient gas exchange. Type I cells may play an important role in lung liquid homeostasis by virtue of their properties of extremely high water permeability and capability of transporting ions. Type II cells, which cover the remainder of the alveolar surface, synthesize, secrete, and recycle surfactant components, have the capacity to transport ions, synthesize immune effector molecules, and act as progenitor cells following injury to the alveolar epithelium. Our response to this challenge grant focuses on the development of two types of reagents: 1) probes for the generation of conditional knockouts specific to either type I or type II cells in the lung that will also confer the ability to FACS sort these cells;and 2) characterization of protein antigens recognized by specific to human type I or type II cells for the purpose of FACS sorting human alveolar epithelial cells and for developing more robust assays to study progression of lung injury and other diseases that damage the alveolar epithelium. There are two specific aims: 1) To generate transgenic animals that can be used for conditional knockouts specifically in alveolar epithelial type I or type II cells and for FACS sorting of the same cells. 2) To develop improved reagents that identify type I or type II cell-specific apical plasma membranes that are suitable for FACS sorting of human alveolar epithelial cells and for use as biomarkers in evaluating disease severity and progression in various states of lung injury. The proposed studies are natural extensions of previous work in our laboratory. The funds available from the TARP mechanism would enable us to proceed in both of these important areas of investigation to produce novel reagents for the study of both lung alveolar epithelial cell biology and human lung disease. PUBLIC HEALTH RELEVANCE: Our response to this challenge grant focuses on the development of two types of reagents: 1) probes for the generation of conditional knockouts specific to either type I or type II cells in the lung that will permit simultaneous FACS sorting of cells;and 2) characterization of protein antigens recognized by antibodies specific to human type I or type II cells for the purpose of FACS sorting human alveolar epithelial cells and for developing more robust assays to study progression of lung injury and other diseases that damage the alveolar epithelium. The proposed studies are natural extensions of previous work in our laboratory but are currently unfunded.